pretty sequencing gels

Hiranya Roychowdhury hroychow at NMSU.EDU
Mon Nov 17 18:49:15 EST 1997


At 11:21 AM 11/17/97 -0800, Wood Lab wrote:
>Help!  I have been trying to generate a sequencing gel that is good 
>enough for publication.  Although the sequence I get is readable, there 
>always appears to be one thing or another preventing top quality.  I am 
>using 33P-labeled ddNTP's and cycle sequencing. <snip>
>
>

I had no idea that people still publishing pictures of seq. gels.
Anyway, bleading, fuzziness etc. may be caused by glass plates that are not
cleaned thoroughly. For 8% gels (regular) we use 50uL of TEMED and 250uL of
20%APS for 100mL solution. We also weigh out Acryl-bis fresh every time
since there would be weeks without any seq. gels to be run. (hence no point
in making stock soln.). Also remember to filter the gel soln. through a
0.45u filter. It may also help to filter the running buffer. 
        The smiling of the bands and fuzziness may alsobe due to uneven
heating of the gel, which is mostly an inherent problem of the rig. Little
can be done about that.  


Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu




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